Abstract 1099: High-throughput engineering and functional annotation of cancer fusion genes

Abstract

Abstract Next generation sequencing (NGS) technologies are rapidly being incorporated into the clinic to facilitate decisions on cancer patient care. Recognizing this, large-scale efforts by The Cancer Genome Atlas (TCGA) and others are generating a compendium of genomic aberrations found across major cancer types with the goal of identifying new therapeutic targets. The challenge now is to find ways to identify functional “driver” aberrations, as targeting driver events or their activated pathways offers the greatest hope of improving patient outcomes. Oncogenic transcript fusions resulting from chromosomal rearrangements represent an important class of such events. The successful targeting of fusion oncoproteins such as BCR-ABL and EML4-ALK with imatinib and crizotinib, respectively, provide strong rationale for comprehensive testing of cancer fusion genes. Unfortunately, the functional interrogation of fusion genes is complicated by the large quantity identified, inability to accurately predict those with driver activity, and significant technical roadblocks preventing fusion gene construction for biological assays. To circumvent these bottlenecks, we developed novel technologies permitting (1) high-throughput fusion gene construction using a novel multi-fragment DNA recombineering strategy with our platform of >35,000 human open reading frame gene clones, and (2) rapid lentiviral delivery of fusion genes to generalized and context-specific cell models to identify those with in vitro and in vivo driver activity and responsiveness to available therapeutics. As proof-of-concept, we used this approach to engineer known fusion oncogenes (BCR-ABL, EML4-ALK, and ETV6-NTRK3) and validated their transforming ability using our in vitro and in vivo driver screening systems, demonstrating our ability to rapidly deliver fusion genes with functional activity. In a pilot screen of fusion genes identified in pan-negative melanoma (i.e., devoid of signature mutations in BRAF and NRAS) by TCGA, we identified multiple uncharacterized BRAF and RAF1 fusion events among others that strongly activate MAPK signaling and exhibit potent transforming activity. Moreover, cells carrying the BRAF and RAF1 gene fusions revealed a marked sensitivity to RAF and MEK inhibitors, a finding that may indicate use of these agents for patients whose tumors harbor these events. We are now scaling these efforts for the comprehensive analysis of uncharacterized gene fusions, ultimately allowing functionalization of thousands of fusion events across diverse cancer types. These systems will reveal the highest priority fusion gene targets to enroll in deep mechanistic biology studies, drug discovery and development programs ultimately leading to personalized treatment strategies. Citation Format: Hengyu Lu, Angeliki Pantazi, Turgut Dogruluk, Armel Dogruluk, Chad Creighton, Gordon B. Mills, Raju Kucherlapati, Kenneth L. Scott. High-throughput engineering and functional annotation of cancer fusion genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1099. doi:10.1158/1538-7445.AM2015-1099